title: Duplex real-time PCR for rapid simultaneous detection of Batrachochytrium dendrobatidis and Batrachochytrium salamandrivorans in Amphibian samples.
abstract: Chytridiomycosis is a lethal fungal disease contributing to declines and extinctions of amphibian species worldwide. The currently used molecular screening tests for chytridiomycosis fail to detect the recently described species Batrachochytrium salamandrivorans. In this study, we present a duplex real-time PCR that allows the simultaneous detection of B. salamandrivorans and Batrachochytrium dendrobatidis. With B. dendrobatidis- and B. salamandrivorans-specific primers and probes, detection of the two pathogens in amphibian samples is possible, with a detection limit of 0.1 genomic equivalent of zoospores of both pathogens per PCR. The developed real-time PCR shows high degrees of specificity and sensitivity, high linear correlations (r(2) > 0.995), and high amplification efficiencies (>94%) for B. dendrobatidis and B. salamandrivorans. In conclusion, the described duplex real-time PCR can be used to detect DNA of B. dendrobatidis and B. salamandrivorans with highly reproducible and reliable results.
authors.affiliation: Department of Pathology, Bacteriology, and Avian Diseases, Faculty of Veterinary Medicine, Ghent University, Merelbeke, Belgium.
authors.forename: M
authors.initials: M
authors.lastname: Blooi
authors.forename: F
authors.initials: F
authors.lastname: Pasmans
authors.forename: J E
authors.initials: JE
authors.lastname: Longcore
authors.forename: A
authors.initials: A
authors.lastname: Spitzen-van der Sluijs
authors.forename: F
authors.initials: F
authors.lastname: Vercammen
authors.forename: A
authors.initials: A
authors.lastname: Martel
mesh_terms: D000663:Amphibians / Q000382:microbiology*; D000818:Animals; D008411:Chytridiomycota / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification*; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods*; D009181:Mycoses / Q000175:diagnosis / Q000382:microbiology / Q000662:veterinary*; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity